Dottorato in SCIENZE FARMACEUTICHE

Titolo della tesi: CRITERIA AND METHODS FOR MICROPLASTICS MONITORING IN WATERS TO BE USED FOR HUMAN CONSUMPTION WITHIN THE NEW EU LEGISLATION FRAMEWORK

Microplastics are ubiquitous contaminants and their release into the environment has long been documented, especially in the last few years. Their widespread presence in several environmental and biological matrices, even in those usually less prone to contamination, have led the regulatory authorities, the scientific community and interested stakeholders to question about possible effects on human health. While the toxicity posed by plastics and microplastics (especially those > 300 µm) to marine organisms seems evident, effects induced by smaller particles on humans are yet to be understood. The main challenge in the identification and toxicological assessments of microplastics rises from the complexity and heterogeneity of these compounds. Regarding microplastics analysis, several different approaches are available. However, the application of different sampling, pre-treatment and analytical methods has resulted in not directly comparable data. In addition, as a consequence of toxicological findings, analytical criteria and matrices investigated are changing. To deal with the increasing levels of plastics and microplastic pollution, the European Commission is adopting a number of actions under the European Green Deal and the Circular Economy Action Plan. In addition, in Directive (EU) 2020/2184 on the quality of water intended for human consumption microplastics appeared for the first time. Described as emerging compounds, microplastics have been related to the “watch list” mechanism introduced with Directive (EU) 2020/2184. The European Commission, by 2024, aims to adopt an analytical method to measure microplastics and, by 2019, to submit a report of risk analysis related to microplastics in drinking water. In order to identify a suitable analytical methodology, the European Commission's Joint Research Centre (JRC) launched a dedicated project in order to harmonize experience and knowledge about microplastic analysis in drinking water, requiring support from national technical-scientific representatives, industry experts and stakeholders. This project includes an online survey and a (series of) workshops designed to collect contribution from stakeholders and experts in microplastics analysis. It is within this complex framework that the present PhD project was structured. This project has multiple purposes, equally divided between the institutional and experimental activities. Institutional activities included the establishment of the Italian National working group on microplastics in drinking water and the discussion on data resulting from the first national survey, based on feedbacks by working group members. The Italian National working group which includes experts from the National Research Council (CNR), national and local environmental Authorities (SNPA: ISPRA and ARPA), Universities and Federation of water suppliers (Utilitalia). The group was designed to work on: (i) JRC and EC support on national expertise about microplastic monitoring in drinking water (ii) development of national analytical method for microplastic in drinking water to be presented to the JRC. From survey data, emerged that experts are flexibly adapting to new challenges posed by microplastics. Regarding experimental activities, specific aims were to develop a method for microplastic analysis suitable for both surface water and water to be used for human consumption, by comparing sampling and analytical techniques, in order to evaluate their pros and cons with a view to a routine approach. Surface water to be used for human for human consumption from the three longest Italian rivers (Po, Adige and Tiber) and water from Drinking Water Treatment Plants (DWTPs) were sampled. For microplastics sampling, a new sampling method, including 2-steps in situ filtration was developed in order to collect several litres of surface water. Assembled filtration system proved to be effective in filtering high volumes of surface water without clogging (average of 1.803,6 L). Tests to assess the goodness of this sampling method for drinking water were also carried out and an average of 2792,6 L litres was filtered without clogging. In order to compare sampling techniques, also water was collected by filling 2.5 L bottles (discrete sampling). Sample pre-treatment and Micro-FTIR analysis were carried out at Istituto di Scienze Polari – Consiglio Nazionale delle Ricerche (ISP-CNR) in Mestre while Micro-Raman analysis were carried out at Department of Chemistry of Padua University. Regarding sample pre-treatment, a “mild” oleo-extraction and purification method was employed. The method has been proved to be adaptable to surface water and efficient to extract microplastics, minimizing any interferers for the analysis. Abundance and polymer identifications were evaluated following the “semi-automated analysis: particle measuring” and “subsampling” approaches in Micro-FTIR. Each IR transmittance spectrum of suspected plastics was then compared with specific microplastics reference libraries. Microplastics were counted, identified and divided by size and shape. For particles between 20 and 100 µm a size range distribution was also performed. Microplastics were observed in every surface water samples collected with both sampling methods, with the exception of the one performed at DWTP #1 with the filtration system, due to inability to properly apply particle measurement to the filters. Microplastics found greatly differed in type and number at each sampling site, while for size distribution and shape greater homogeneity was observed. In terms of polymer composition, heterogeneity was observed, probably due to differences between the various sampling sites. In samples from the filtration systems, polyester, fluorocarbon, polytetrafluoroethylene and acrylic were retrieved in both samples while, in their corresponding discrete samples, only fluorocarbon and polyolefin were common to all sites. Thus, only fluorocarbon was common to all surface water samples. A higher homogeneity was noted by comparing the composition of plastics in filtration system samples with the corresponding discrete samples of the same site. However, only 5 polymers out of 9 (approx. 56%) were common in DWTP #2 and only 5 polymers out of 12 (approx. 42%) were common in DWTP #3. These differences underline the necessity of performing also studies on sampling methods when developing protocols for microplastics analysis, as results may be very different by changing the sampling input. In terms of size, a higher homogeneity was observed. Only approximately 5% of particles found was bigger in size than 100 µm (max 515,9 µm) and approximately 95% of particle size varied between 20 and 100 µm. In any case, particles between 7 µm (LoD) and 20 µm, potentially the most harmful ones, were not retrieved in any samples analyzed with Micro-FTIR. In addition, a detailed dimensional analysis of particles 20 – 100 µm fraction was performed for each sample. Data showed that 40-50 µm size cluster was the most populated for every sample. The 40-50 µm size cluster remained the most populated even if stratifying by sampling techniques and assuming a 1000 L volume for filtration system samples. A general trend in which particles are more condensed in the region to the left of the 60-70 µm cluster can be observed. In terms of shape, a high degree of homogeneity was observed among samples. Non-elongated particles were by far the most common particles (AR < 2) in all samples. The proportion of non-elongated particles over the total remained essentially the same stratifying data for sampling type (approx. 65% for filtration system samples assuming 1000 L as a reference volume and approx. 65% for discrete samples). Raman microscopy analysis was performed on two samples from DWTP #2 and DWTP #3 following the Point and Shoot and Imaging/Mapping approach. Several suspected microplastics were identified with Point and Shoot approach while in Imaging/Mapping a 50x50µm surface area with a 2 µm-spaced grid was mapped. During the Imaging/Mapping, polyvinyl chloride and polyethylene signals (R = 0.53 and R = 0,69), were found in two different points, without any similar signals in the surrounding area. Thus, two suspected microplastics < 2 µm were detected in the sample. Experimental showed that microplastics are present in surface water in significant amounts and have the opportunity to reach DWTPs. Each site showed different polymers composition but small microplastics (SMPs) (especially those non elongated and < 70 µm) proved to be the most abundant group.